The objective of this proposal is to isolate and characterize a potent growth factor that exists in the mammalian testis. This mitogenic polypeptide will induce DNA synthesis and cell division in confluent, quiescent Balb/c 3T3 cells and sparsely-plated prepuberal Sertoli cells. Most of the mitogenic activity is concentrated in prepuberal Sertoli cells, to a lesser extent in primitive type A spermatogonia and preleptotene spermatocytes, but none is detected in germ cells at any other stage of differentiation. An homogenate of prepuberal Sertoli cells at 20 micrograms/ml protein induces half-maximal DNA synthesis in confluent 3T3 cells. Maximal stimulation results in a 70-l00-fold increase in (3H) thymidine incorporation into DNA, labeling of greater than 99% of 3T3 cell nuclei, and a doubling in cell number over 48 hrs. The growth factor is sensitive to proteases and heating at l00 degrees Centigrade for 2 min, but not to dithiothreitol. Gel filtration chromatography reveals that the polypeptide has a M.W. of approximately 16,000. Considerable effort will be directed toward purifying the polypeptide to homogeneity, using standard techniques such as gel filtration and hydrophobic chromatography, preparative isoelectric focussing, SDS-polyacrylamide gel electrophoresis and affinity chromatography. The successive purification steps are to be monitored by assaying the specific mitogenic activity and by SDS-PAGE. The pure polypeptide will be characterized biochemically to provide amino acid composition, amino- and carboxyl-termini and carbohydrate content. Furthermore, monoclonal antibodies directed specifically against the growth factor will be prepared using the hybridoma technique.